Various attempts have been made to microencapsulate biologically-active macromolecules, tissue and individual cells so that they remain viable and in a protected state within a semi-permeable membrane which permits passage of low molecular weight substances, such as nutrients and oxygen, but not of high molecular weight substances, such as, proteins and cells. However, none of these attempts has been successful in providing microcapsules in which tissue or cells enclosed within the semi-permeable membrane are able to survive in an animal body for longer than 2 to 3 weeks, which severely limits the utility of the products in the treatment of diseases requiring organ transplantation, such as diabetes.
In "Semipermeable Microcapsules" by T. M. S. Chang, Science, 146, 1964, 524 to 525, there is described the microencapsulation of erythrocyte hemolysate and urease in semi-permeable polyamide (nylon) membranes. These microcapsules did not survive for long when injected into the blood stream. Papers have described the preparation of semi-permeable microcapsules containing microbial cells and viable red blood cells, namely K. Mosbach and R. Mosbach, Acta Chem. Scand., 20, 1966, 2807 to 2812 and T. M. S. Chang, F. C. MacIntosh and S. G. Mason, "Semi-permeable Aqueous Microcapsules", Can. J. Physiol. and Pharmacology, 44, 1966, 115 to 128. The Chang et al article mentions for the first time the possibility of using injections of encapsulated cells for organ replacement therapy.
The next significant development was the use of calcium and aluminum alginate gels for the immobilization of microbial cells and enzymes. The cells were immobilized under extremely mild conditions, thus maintaining their viability. This work was described in V. Hackel, J. Klein, R. Megret and F. Wagner, Europ. J. Appl. Microbiol., 1, 1975, 291 to 296 and M. Kierstan and C. Bucke, "The Immobilization of Microbial Cells, Subcellular Organelles, and Enzymes in Calcium Alginate Gels", Biotechnology and Bioengineering, 19, 1977, 387 to 397.
Subsequently, viable tissue and cells were immobilized in alginate droplets coated with polylysine (F. Lim and R. D. Moss, "Microencapsulation of Living Cells and Tissues", J. Pharm. Sci. 70, 1981, 351 to 354). While the cells remained viable in culture for up to two months, no experiments are described to test the in-vivo biocompatibility of the polylysine membrane. At approximately the same time, there was reported for the first time, the use of microencapsulated islets to correct the diabetic state of diabetic animals, in F. Lim and A. M. Sun, "Microencapsulated Islets as Bioartificial Pancreas", Science, 210, 1980, 908 to 909. However, the microcapsules, consisting of an inner alginate core, followed by a polylysine coat and an outer polyethyleneimine membrane, were rejected by an animal body within 2 to 3 weeks of implantation due to the poor biocompatibility of the outer polyethyleneimine membrane.
Formation of the latter microcapsules also is described in U.S. Pat. No. 4,352,883 F. Lim. As set forth therein, finely divided living tissue is suspended in an aqueous medium which contains sodium alginate, the suspension is formed into droplets of a size to envelop tissue, the droplets are gelled by conversion to calcium alginate to form discrete, shape-retaining temporary capsules, a permanent semi-permeable membrane is formed about the temporary capsules, and the calcium alginate gel is reliquified within the membrane by ion exchange. Example 3 of the patent describes injection of the microcapsules into diabetic rats.
In the Lim patent, it is indicated that, proteins or polypeptide cross-linkers, such as, polylysine, are readily attacked in vivo resulting in rapid destruction of the membrane whereas cross-linkers which are not readily digestible in mammalian bodies, result in longer lasting membranes. Example 1 of the Lim patent discloses the use of polyethyleneimine and polylysine to form the semipermeable membrane and the article of Lim and Sun mentioned above shows that such microcapsules do not possess an in vivo life of more than two to three weeks.
The parent application Ser. No. 953,413 of the application which lead to the Lim patent contains an Example 1 which uses polylysine as the sole membrane polymer. Although in vitro test results are given, no in vivo studies are specified. No reliquification step is specified in this Example.
The Lim patent discloses that it may be desirable to treat the microcapsules so as to tie up free amino groups which otherwise would impart to the capsules a tendency to clump, by immersing the capsules in a solution of sodium alginate.
Example 1 of the patent discloses immersion of the microcapsules in alginic acid solution. Example 1 of the parent application discloses immersion of the microcapsules in a 0.1% sodium alginate solution. The capsules can be engineered to have a selected in vivo useful life by astute selection of the cross-linking polymer. Proteins or polypeptide crosslinkers, e.g., polylysine, are readily attacked in vivo resulting in relatively rapid destruction of the membrane. Cross-linkers not readily digestible in mammalian bodies, e.g., polyethyleneimine, result in longer lasting membranes." As is apparent from the results achieved by Lim and Sun referred to above using microcapsules comprising membranes including polyethyleneimine, the organ replacement therapy lasts for a period no longer than about three weeks.
U.S. Pat. No. 4,352,883 mentions the possibility of using polylysine, a much more biocompatible material, instead of polyethyleneimine as the membrane. Polylysine is positively charged and it is well known that positively-charged surfaces are excellent substrates for cell growth. Cell growth on the surface of the microcapsules, such as would occur with a polylysine membrane, would transform the semipermeable capsular wall to an impermeable one, resulting in the death of the encapsulated tissue.
It is apparent, therefore, that there is a need for the development of microcapsules which can be implanted into an animal body and be effective in the treatment of diseases requiring organ transplantation, such as, diabetes, for extended periods of time.